1. Technical Field
The present invention relates to pharmaceuticals, and especially radiopharmaceuticals for use as diagnostic and therapeutic agents. More specifically, the present invention relates to compounds and methods of synthesizing compounds which utilize both mono- and multi-dentate ligands which form stable complexes with metal compounds without the need for external reducing agents for use as diagnostic or therapeutic radiopharmaceuticals.
2. Background Art
Because of the favorable physical properties, widespread availability, and low cost of .sup.99m Tc, this radionuclide continues to be the most attractive candidate to formulate diagnostic radiopharmaceuticals for scintigraphic imaging studies in patients (Jurisson et al., 1993). Re, a chemical analogue of Tc, has two radioisotopes (i. e., .sup.186 Re and .sup.188 Re; .sup.186/188 Re) that have physical and production properties that make them among the most attractive beta-emitting radionuclides for formulation of new therapeutic radiopharmaceuticals (Volkert et al., 1991; Troutner, 1987). .sup.105 Rh is another important beta-emitting radionuclide for use in preparing therapeutic radiopharmaceuticals (Troutner, 1987). Since the chemical properties of Tc and Re are often identical (although, not always) many ligand systems can be used as a basis to synthesize bifunctional chelating agents (BFCAs) that are capable of forming chelates with .sup.99m Tc that have the same structural and physicochemical properties as the corresponding .sup.186/188 Re chelates.
Development of sophisticated molecular probes in the design of new .sup.99m Tc-, .sup.186/188 Re, and .sup.105 Rh radiopharmaceuticals will provide for future advances in the diagnosis and treatment of patients. While many important single photon emission computed tomography (SPECT) radiopharmaceuticals are effectively used as specific tools for diagnosis of human disease, accelerated development of many new site-directed synthetic derivatives (e.g., immunologically derived molecules, receptor-avid molecules, etc.) will provide a multitude of opportunities for further technological advances for both diagnostic and therapeutic applications. Many difficulties encountered in the design of highly selective radiolabeled drug carriers must be overcome (e.g., problems in efficient drug delivery to target sites, in vivo metabolism, rates of clearance of radioactivity from non-target tissues relative to target tissues, etc.). The physicochemical characteristics of the .sup.99m Tc-, .sup.105 Rh-, and .sup.186/188 Re-chelate moiety attached or fused to the site-directed molecule will play a crucial role as an inherent determinant of the effectiveness of the final drug product. In addition, the ability of .sup.99m Tc or .sup.186/188 Re to label the final product under conditions amenable for routine formulation of radiopharmaceuticals is also an essential consideration.
Labeling of biomolecules with .sup.99m Tc or .sup.186/188 Re to produce effective radiopharmaceuticals presents many challenges. It is necessary to produce .sup.99m Tc and/or .sup.186/188 Re labeled drugs that have high in vitro and in vivo stabilities. Several different ligand frameworks have been developed that form .sup.99m Tc or Re chelates exhibiting minimal or no measurable in vivo or in vitro dissociation. These chelates have provided radiopharmaceutical chemists with a selection of .sup.99m Tc-chelates that have a range of physicochemical characteristics. The formation of .sup.99m Tc (viz Re) products in high yields with high radiochemical purity (RCP), however, usually requires the presence of large quantities of excess ligand during the formulation processes that are used for routine pharmaceutical preparation. Unfortunately, the high specific activities (i.e., GBq/.mu.mole or Ci/.mu.mole) required for radiolabeled site-directed synthetic derivatives being developed precludes the use of many of these chelation systems, thus, severely limiting the choice to only a few ligand backbones.
High specific activity (Sp. Act) radiolabeled agents can be prepared using either preformed .sup.99m Tc- or .sup.186/188 Re bifunctional chelates (BFCs) or post-conjugation chelation with the radioactive metals where a chelating moiety is already appended (Parker, 1990) or fused (Lister-James et al., 1994; Knight et al., 1994) to the biomolecular targeting agent. Even though maximization of Sp. Act can be achieved by separation of the radiolabeled from the non-radiolabeled molecules, practically it is more desirable to employ chelation systems that require small quantities of the chelates. In the formation of products that will be ultimately used as FDA approved .sup.99m Tc/.sup.186/188 Re radiopharmaceuticals for routine patient care applications, it is most desirable to keep the number of steps for the formation of the drug-product to a minimum, ideally to one step, as is the case for most .sup.99m Tc- "instant kits".
One of the few ligand systems shown to be effective for preparation of high yield, stable .sup.99m Tc chelates using small quantities of chelator are the amido-thiol class of ligands (Fritzberg et al. 1988, Rao et al., 1992, and Chianelli et al, 1994). Generally, these types of multi-dentate ligands contain at least four donor atoms and one or two thiol donor groups in combination with 2-3 amido donor groups. Several N.sub.2 S.sub.2 or N.sub.3 S amido-thiol frameworks have been used to synthesize BFCAs and include diamido-dithiol (DADS) ligands (Fritzberg et al., 1988), monoaminemonoamide (MAMA) ligands (Rao et al., 1992; Gustavson et al., 1991) and mecaptoacetylglycylglycyl-glycine (MAG.sub.3) ligands (Chianelli et al., 1994). While the amido-thiol ligands make effective BFCAs for .sup.99m Tc and .sup.186/188 Re, the range of their physicochemical properties are limited, conditions for routine labeling can be difficult to reduce to practical utility and external reducing agents (e.g., Sn(II) are usually present during labeling with .sup.99m Tc or .sup.186/188 Re, which can cause irreversible alteration of the site-directed moiety reducing or eliminating specific in vivo localization.
Other ligand systems that have also been used for .sup.99m Tc labeling include N.sub.2 S.sub.2 -amine-thiol ligands, propylineamineoxime (PnAO) derivatives and the hydrazino nicotinamide (HYNIC) system. The former two derivatives form neutral lipophilic .sup.99m Tc-chelates, that while beneficial in some respects, result in high non-specific binding in vivo and poor clearance from non-target tissues (Muna et al., 1994; Noch et al., 1994). The HYNIC system does not form a well-defined product with .sup.99m Tc (Abrams et al., 1990a; Abrams et al., 1990b). All of these systems usually form chelates with .sup.99m Tc with the necessity of external reducing agents.
Ligand backbones containing trivalent phosphine donor groups have been shown to be effective in forming stable .sup.99m Tc and .sup.186/188 Re chelates in high RCP. Phosphines not only chelate .sup.99m Tc (or Re), but they are capable of reducing both pertechnetate and perrhenate to lower oxidation states, and, therefore, do not necessarily require the presence of an external reducing agent e.g., Sn(II)!. Diphosphine ligands have been extensively used in the development of .sup.99m Tc-radiopharmaceuticals, particularly those that are used as .sup.99m Tc-labeled myocardial perfusion agents (Deutsch, 1993; Nowotnik and Nunn, 1992; Kelly et al., 1993). Unfortunately, most of these chelates utilize alkyl-phosphine donor groups and the phosphines are rapidly oxidized (to phosphorus oxides) in aqueous solutions containing O.sub.2 and require stringent conditions for manufacture of the drugs and for ultimate routine formation of the final product. For these reasons, ligands that contain alkyl phosphine donor groups have limited flexibility for the design of new drugs and do not form a rational basis to prepare most phosphine-based BFCAs for use in preparing site-directed radiopharmaceuticals. Aromatic phosphines have also been reported for use with Tc and Re, however, the high lipophilicity of the resulting chelates minimize their potential utilization as BFCAs for in vivo applications.
A small ligand system containing phosphine donor groups with good solubility in aqueous solutions and not oxidized by O.sub.2, but still capable of reducing .sup.99m Tc0.sub.4.sup.- or .sup.186/188 ReO.sub.4.sup.- and/or strongly chelating reduced Tc or Re, would find widespread applicability in formulating new radiopharmaceuticals or new BFCAs.
Most other bifunctional chelation systems require the presence of an external reducing agent (e.g., Sn.sup.+2) or prereduction of .sup.99m Tc0.sub.4.sup.- or .sup.186/188 ReO.sub.4.sup.- to lower metal oxidation states (e.g. .sup.99m Tc-glucoheptonate). Water soluble phosphine groups containing low molecular side arms attached to each phosphine P-atom would provide versatility in ligand design and could be used as both as a reducing agent for .sup.99m Tc0.sub.4.sup.- (or .sup.186/188 ReO.sub.4.sup.-) under conditions used for routine .sup.99m Tc-radiopharmaceutical preparation and as an efficient complexing agent for the reduced forms of Tc or Re.
Applicants use a mono-dentate phosphine ligand and a series of multi-dentate ligands containing functionalized hydroxyalkyl phosphines that are stable in aerated aqueous solutions and will form highly stable .sup.99m Tc and .sup.188 Re chelates. Unlike prior art alkyl phosphine based ligands designed to reduce or chelate .sup.99m Tc or .sup.186/188 Re, the hydroxyalkyl phosphine groups are not sensitive to the presence of oxygen when dissolved in aqueous solutions. Other water soluble phosphine ligands with good oxidative stability have also been used as reducing agents, however, the side chains attached to the phosphine donor P-atoms in these ligands are bulky and produce highly charged phosphines which limit their utility in radiopharmaceutical development (Pasqualine et al., 1994).
Most other bifunctional chelation systems require the presence of an external reducing agent (such as Sn(II) or NaBH.sub.4) or prereduction in order to reduce the .sup.99m Tc0.sub.4.sup.- (or .sup.186/188 ReO.sub.4.sup.-) from the +7 oxidation state to lower oxidation states (e.g., .sup.99m Tc-GH) that are more readily chelated.
The ligands containing one or more hydroxyalkyl phosphine donor groups of the present invention require no external reducing agents, however, the ligand can be used as coordinating groups when used in conjunction with other reducing agents or .sup.99m Tc-synthons. The resulting .sup.99m Tc and Re complexes produced with these phosphine containing ligands exhibit excellent in vivo stability as well in aqueous solutions including human serum.